Compounds and uses thereof for treating inflammation and modulating immune responses

ABSTRACT

The present invention provides compounds, and compositions comprising these compounds, which have immunomodulatory activity and/or anti-inflammatory activity.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 61/143,925, filed Jan. 12, 2009, which is incorporated herein byreference in its entirety, including all figures, tables and sequences.

BACKGROUND OF THE INVENTION

In response to injury, cancer, microbial invasion, and the like, humansmount inflammatory reactions to control the pathological condition andto initiate a repair process. During inflammation, various immune cellsincluding T-lymphocytes, neutrophils and macrophages are recruited tothe site of infection and produce cytokines to facilitate the immuneresponse. Among these cytokines, tumor necrosis factor-α (TNF-α) is oneof the major proinflammatory proteins to mediate the immune defense.

In addition to acute phase response, TNF-α has been shown to be involvedin the progression of various chronic diseases including tumorigenesisand rheumatoid arthritis (RA). The dysregulation of TNF-α production wasdemonstrated to be involved in different stages of tumorigenesisincluding initiation of tumor growth¹, cell proliferation² andinvasion³. For tumor cell proliferation, TNF-α upregulates specificgrowth factors to mediate the malignant growth. The cytokine promotesangiogenesis favoring growth of blood vessels to support the tumormigration, and thus plays a key role in tumor metastasis. For example,glioblastoma migration and induction of metalloproteinases aresignificantly enhanced in response to TNF-α effects⁴.

Examples of chronic disease pathogenesis mediated by TNF-α includerheumatoid arthritis and inflammatory bowel diseases. Patients withrheumatoid arthritis have a low grade insidious inflammation in thesynovial tissues. It is known that overproduction of TNF-α at theinflamed joint leads to slow destruction of the joint cartilage andsurrounding bone.

During an acute phase of infection such as in the case of sepsis,uncontrolled production of TNF-α is well known to cause deleteriouseffects to the host. Sepsis is the second most common cause of death innon-coronary intensive care units and the tenth leading cause of deathoverall in high-income countries⁵. The clinical outcome of infectionleading to sepsis is primarily associated with the excessive stimulationof the host immune cells, particularly monocytes or macrophages, bybacterial endotoxins (e.g., lipopolysaccharide [LPS])⁶⁻⁸. Macrophagesoverstimulated by LPS also produce high levels of mediators such asinterleukin-1 (IL-1), IL-6, and TNF-α⁹. These mediators are implicatedin the pathogenesis of sepsis and found to be contributing factors tothe demise of the host. The development of novel therapies directedtowards the inhibition of TNF-α production may help to aid in thetreatment of these acute and chronic diseases described above.

Following exposure to pathogens and endotoxins, intracellular signalingpathways including specific kinases and transcription factors areactivated to induce the expression of TNF-α. The involvement ofmitogen-activated protein (MAP) kinases and the nuclear factor kappa B(NF-κB) in pathogen-induced TNF-α expression are well documented¹⁰⁻¹² .Mycobacteria, avian influenza and HIV-1 Tat protein are inducers ofTNF-α through the MAP kinases¹³⁻¹⁵.

There are three MAP kinase subtypes including extracellularsignal-regulated kinase-1/2 (ERK 1/2), p38 MAP kinase and c-JunN-terminal kinase (JNK)¹⁶⁻²⁰ known in humans. They transduce a varietyof extracellular stimuli through a cascade of protein phosphorylationsthat lead to the activation of transcription factors such as NF-κB. Theactivation of NF-κB is crucial in production of cytokines including IL-6and TNF-α¹³⁻¹⁵. The process occurs by the phosphorylation of I-κB atSer32 and Ser36 via the I-κB kinase (IKK) signalosome complex followedby proteosomal degradation²¹ and consequent dissociation of I-κB andNF-κB subunits²². The activated NF-κB is then translocated from thecytoplasm to the nucleus, where it binds to KB binding sites in thepromoter region of responsive genes, leading to the initiation oftranscription of pro-inflammatory mediators. Because inappropriateactivation of NF-κB is associated with a wide range of human diseases²³,it has been considered as a plausible target for therapeuticintervention.

Non-steroid anti-inflammatory drugs (NSAIDs) including aspirin,ibuprofen, and indomethacin are well-known in ameliorating acute andchronic pain associated with inflammatory diseases such as rheumatoidarthritis and inflammatory bowel disease. However, they are noteffective in the treatment of advanced stages of rheumatoid arthritisand related autoimmune diseases. For those conditions, steroids andcytotoxic drugs such as methotrexate and cyclophosphamide are used.These drugs are associated with severe adverse effects includinggastrointestinal irritation, severe bleeding, and bone marrowsuppression.

In recent years, immunotherapeutics have been developed which aim at theneutralization of TNF-α and suppression of its undesirableproinflammatory effects. These include soluble TNF-α receptor (Enbrel)and anti-TNF-α antibody (Infliximab). Despite their novelty and efficacyin the arrest of disease progression, they are very expensivetherapeutic regimens.

Considerable effort has been made in efforts to discover bioactiveagents from natural sources, especially from microbes, plants, andmarine organisms. Plants act as an alternative and supplemental sourceof new medicine, as they contain a variety of previously unknownchemicals that may have potent biological effects.

Traditional Chinese medicine has been practiced by the Chinese peoplefor 2-3 millennia. It deals with pathology, and diagnosis, treatment andprevention of diseases. Chinese medicinal materials have been recordedin various pharmacopoeia. One of the classical references for medicinalherbs is Ben Cao Gang Mu written by Li Shizhen in the late 14^(th)Century. The book contains about 2,500 items of herbs and other productsincluding animals and minerals.

Herbs used in traditional Chinese medicine are commercially available.Common herbs include Ren Shen (Ginseng radix), Gang Gui (Angelicasinensis radix), Huang Qi (Astragali radix), Gan Cao (the rhizome ofglycyrrhiza uralensis Fisch., Glycyrrhiza glabra L. or Glycyrrhizainflata Bat, and preferably Glycyrrhiza uralensis Fisch), and Huang Qin(Scutellariae radix). Commonly, herbs are obtained in their dry forms,sometimes already grinded into powder.

Cimicifuga rhizome has a long and diverse history of medicinal use inthe Eastern United States and Canada²⁶. Historically, native AmericanIndians used it to treat a variety of conditions including malaise,malaria, rheumatism, abnormalities in kidney function, sore throat,menstrual irregularities, and menopause²⁶⁻²⁸. In Asian countriesincluding China, Japan and Korea, Cimicifuga racemosa and itscounterparts Cimicifuga heracleifolia, Cimicifuga foetida and Cimicifugadahurica have been used as traditional medicinal herbs to treat fever,pain and inflammation²⁹⁻³⁰.

Previous studies demonstrated the inhibitory effects of Cimicifugaracemosa extract on histamine, bradykinin and cyclooxygenase-2 (COX-2)mediated inflammatory actions³¹. The extract also has protective effectsagainst menadione-induced DNA damage through its scavenging effects onreactive oxygen species³². In addition, Cimicifuga heracleifoliaextracts has been demonstrated to have anti-viral activities againstrespiratory syncytial virus³⁰. In a recent study, Cimicifuga foetidaextracts were shown to induce apoptosis and cell cycle arrest ofhepatocarcinoma cells, which are critical effects in inhibiting thetumor progression³³. Also, the actions of Cimicifuga racemosa onmenopause-regulated response have been well studied³⁶. These dataindicate that the constituents of Cimicifuga racemosa might functionsimilar to that of estrogen. Other studies showed that Cimicifugaracemosa perturbs cytokine signaling in order to mediate otherbiological functions³⁷.

Currently, in the treatment for rheumatoid arthritis, psoriasis,psoriatic arthritis and ankylosing spondylitis, monoclonal TNF-αantibody plays an important role in the control of disease progression.Similarly, several randomized, double blind, placebo-controlled clinicaltrials had been performed in patients with Crohn's disease. The resultsof these clinical trials showed that the anti-TNF-α antibody(Infliximab) has beneficial effects to the patients⁴¹.

Additionally, recent studies showed that inflammatory responsesincluding TNF-αproduction may play an important role in the pathogenesisof cardiovascular diseases (CVD). It has been suggested that TNF-α maydestabilize the atherogenesis and atherosclerotic plaques leading totheir rupture, resulting in myocardial infraction or stroke in CVDpatients.

During microbial infection, macrophages are activated to producecytokines to mediate immune response. Depending on the invading microbeand its biological properties, the host immune system utilizes differentsets of cytokines to combat the invading pathogen locally andsystemically.

A good example is mycobacterial infection, in which the proinflammatorycytokines TNF-α plays a critical role in host survival by propagatinginflammation to contain the microbes by the formation of granuloma⁴².The protective role of TNF-α in controlling mycobacterial growth isexemplified by the resurgence of tuberculosis in patients receivinganti-TNF-α antibody therapy⁴³.

Although the effects of proinflammatory cytokines are protective, theiroverproduction may have adverse effects to the host. In fact,uncontrolled induction of proinflammatory cytokine can lead tocomplications such as hypotension, organ failure and even death^(44,45).Indeed, the overproduction of TNF-α in endotoxemia patients leads toserious deleterious symptoms. In chronic diseases such as rheumatoidarthritis, TNF-α overexpression is known to be the damaging factor andis associated with progressive joint destruction⁴⁶.

BRIEF SUMMARY

The present invention provides compounds, and compositions comprisingthese compounds, which have immunomodulatory activity and/oranti-inflammatory activity. In certain embodiments, because of theeffects of these compounds on TNF-α, they have immunomodulatory activitythat is not specifically associated with inflammation.

One embodiment of the subject invention pertains to a compound isolatedfrom herbs. Advantageously, this compound possesses potentanti-inflammatory and immunomodulatory effects.

The present invention thus relates to a substantially pureanti-inflammatory compound having the following structure:

-   -   wherein    -   R₁ is alkyl;    -   R₂ is H or alkyl;    -   R₃, R₄, and R₅ are independently —H, acyl, halo, haloalkyl,        amino, alkylamino, hydroxyl, alkyl, hydroxylalkyl, or —COOH;    -   R₆ is —O or —NH;    -   R₇ is —H, alkyl, alkoxy, hydroxylalkyl, hydroxyl, or halo;    -   R₈, R₉, and R₁₂ are independently —H, acyl, halo, amino,        alkylamino, hydroxyl, alkyl, hydroxylalkyl, or —COOH;    -   R₁₀ is H or alkyl; and    -   R₁₁ is H or alkyl;

Advantageously, in one embodiment, the compounds of the subjectinvention can inhibit LPS-induced TNF-α production. (AL-to-David pleaseconsider adding this with IP language: Due to its potent inhibition ofendotoxin (LPS) effects, the use of the compounds of the subjectcompounds of the subject invention can be applied beyond endotoxemia toinclude inflammatory conditions found in autoimmune diseases and otherrelated conditions.

The present invention is also directed to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and ananti-inflammatory compound of the invention. In a preferred embodiment,the composition contains the anti-inflammatory compound as the activeingredient.

The present invention is also directed to methods of use of thecompounds or compositions comprising them, for the inhibition ofinflammation in animals, preferably mammals, including humans. Thepresent invention is also directed to methods of use of said compoundsor compositions comprising said compounds for the modulation of immuneactivity in animals, preferably mammals, including humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an extraction scheme of B22EES1-8-3 from Cimicifugaracemosa. Cimicifuga racemosa (1.8 kg) was milled and extracted with 500mL milli-Q water for 1 hr with continuous sonication. The collectedsupernatant was then partitioned with ethyl acetate (EtOAc) (1:1). Theresulting dried EtOAc extract was reconstituted and then sequentiallypartitioned with hexane (n-C₆H₁₄), EtOAc and butanol (n-BuOH). Usingbioassay guided fractionation scheme, the fractions showing inhibitoryeffects on LPS-induced TNF-α production were subjected to silica gel 60A(35-75 μm) chromatography and reversed-phase high-performance liquidchromatography using gradient elution until a single compound withanti-inflammatory activity was obtained.

FIGS. 2A-2B show HPLC chromatogram and UV absorbance of B22EES1-8-3. Thecompound was purified by reversed-phase HPLC using gradient elution from25% to 90% of acetonitrile at a flow rate of 1 mL min⁻¹. (A) A singlepeak was detected using Photo-diode Array detector at 254, 210 and 280nm. B22EES1-8-3 was eluted at approximate 9.4 min. (B) The UV absorbanceof B22EES1-8-3 maximized at 290 and 325 nm which revealed that it had aconjugated aromatic system.

FIG. 3 shows the ¹H (upper panel) and ¹³C NMR (lower panel) spectra ofB22EES1-8-3. The structure of B22EES1-8-3 was elucidated by a Bruker 500MHz DRX NMR spectrometer, operating at 500 MHz for ¹H and at 125.765 MHzfor ¹³C NMR, using methanol-d as the solvent.

FIGS. 4A-4B show a bioassay guided fractionation of Cimicifuga racemosa.Primary blood macrophages (PBMac) were treated with different C.racemosa fractions at 100 μg/mL for 24 hr prior to the addition of 20ng/mL LPS for 3 hr. RT-PCR (A) and quantitative RT-PCR (B) assays ofTNF-α and GAPDH were performed afterwards. The results shown arerepresentative of at least three independent experiments, with cellsobtained from different donors. *P<0.05, compared with the correspondingcontrol.

FIGS. 5A-5B show inhibition of LPS-induced TNF-α production byB22EES1-8-3 and dexamethasone. PBMac were incubated with (A) 140 μMB22EES1-8-3 or (B) 1.3 or 5.1 μM dexamethasone (Dex) for 24 hr prior tothe addition of 1 ng/mL and 10 ng/mL LPS for another 24 hr. The culturesupernatants were collected and assayed for TNF-α by ELISA. The resultsshown were the mean values±standard derivation (S.D.) of 6 independentexperiments, with cells obtained from different donors. *P<0.05,compared with the corresponding control.

FIGS. 6A-6C show the effects of B22EES1-8-3 on LPS-inducedphosphorylation (phospho-) of ERK1/2 and p38 MAP kinases, and nucleartranslocation of NF-κB p65. PBMac were incubated with B22EES1-8-3 (140μM) for 24 h prior to the addition of 10 ng/mL LPS for an additional 15min. Cytoplasmic (A, B) and nuclear (C) proteins were harvested forWestern Blotting: (A) Cytoplasmic proteins: phospho-ERK1/2 and totalERK1/2. (B) Cytoplasmic proteins: phospho-p38 and total p38 kinase. (C)Nuclear proteins: upper panel, NF-κB p65 and lamin B; lower panel, theintensity of corresponding lanes in the gel photograph of NF-κB p65 wasshown. The results shown are representative of at least threeindependent experiments, with cells obtained from different donors.*P<0.05, compared with the corresponding control.

FIGS. 7A-7B show the HPLC chromatograms of CF22EES1-8 (A) and CH22EES1-8(B). Herbs C. foetida and C. heracleifolia were extracted following theextraction procedure of C. racemosa. Their extracts (CF22EES1-8 andCH22EES1-8) were injected into the HPLC using the same condition as thatof B22EES1-8-3 and the chromatograms were recorded. The chromatogramsshowed the presence of a compound (with *) with retention time atapproximate 9.4 minutes.

FIGS. 8A-8C show the HPLC chromatograms and HRESI-MS spectra of (A)B22EES1-8-3, (B) CH22EES1-8, and (C) CH22EES1-8. Herbs C. foetida and C.heracleifolia were extracted following the extraction procedure of C.racemosa. Their fractions (CH22EES1-8 and CH22EES1-8) were injected intoan HPLC-coupled high-resolution ESI-TOF-MS using the same condition asthat of B22EES1-8-3. The chromatograms showed the presence of a compound(with *) with retention time at approximately 6 min and with an ion peakat 357 m/z.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a primer useful according to the subject invention.

SEQ ID NO:2 is a primer useful according to the subject invention.

SEQ ID NO:3 is a primer useful according to the subject invention.

SEQ ID NO:4 is a primer useful according to the subject invention.

DETAILED DESCRIPTION

Novel and advantageous compounds have been identified according to thesubject invention. Advantageously, these molecules have usefulimmunomodulatory and/or anti-inflammatory properties. The presentinvention further provides compositions comprising these compounds aswell as methods for the use in treating inflammatory and immuneconditions in a subject.

One embodiment of the subject invention pertains to a compound isolatedfrom herbs. Advantageously, this compound possesses potentanti-inflammatory and immunomodulatory effects.

The present invention thus relates to a substantially pureanti-inflammatory compound having the following structure:

whereinWherein

-   -   R₁ is alkyl;    -   R₂ is H or alkyl;    -   R₃, R₄, and R₅ are independently —H, acyl, halo, haloalkyl,        amino, alkylamino, hydroxyl, alkyl, hydroxylalkyl, or —COOH;    -   R₆ is —O or —NH;    -   R₇ is —H, alkyl, alkoxy, hydroxylalkyl, hydroxyl, or halo;    -   R₈, R₉, and R₁₂ are independently —H, acyl, halo, amino,        alkylamino, hydroxyl, alkyl, hydroxylalkyl, or —COOH;    -   R₁₀ is H or alkyl; and    -   R₁₁ is H or alkyl;        “Alkyl” means linear saturated monovalent radicals of one to        eight carbon atoms or a branched saturated monovalent of three        to eight carbon atoms. It may include hydrocarbon radicals of        one to four or one to three carbon atoms, which may be linear.        Examples include methyl, ethyl, propyl, 2-propyl, n-butyl,        iso-butyl, tert-butyl, pentyl, and the like.        “Acyl” means a radical —C(O)R where R is hydrogen, alkyl or        cycloalkyl, or heterocycloalkyl. Examples include formyl,        acetyl, ethylcarbonyl, and the like.        “Halo” means fluoro, chloro, bromo, or iodo, such as bromo and        chloro.        “Haloalkyl” means alkyl substituted with one or more same or        different halo atoms, e.g., —CH₂Cl, —CH₂Br, —CF₃, —CH₂CH₂Cl,        —CH₂CCl₃, and the like.        An “amino” is intended to mean the radical —NH₂.        “Alkylamino” means means a radical —NHR or —NR₂ where each R is        independently an alkyl group. Examples include methylamino,        (1-methylethyl)amino, methylamino, dimethylamino,        methylethylamino, di(1-methyethyl)amino, and the like.        A “hydroxy” is intended to mean the radical —OH.        Hydroxyalkyl” means an alkyl radical as defined herein,        substituted with one or more, preferably one, two or three        hydroxy groups. Representative examples include, but are not        limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl,        3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl,        2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl,        2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl,        2,3-dihydroxybutyl, 3,4-dihydroxybutyl and        2-(hydroxymethyl)-3-hydroxy-propyl, preferably 2-hydroxyethyl,        2,3-dihydroxypropyl and 1-(hydroxymethyl)2-hydroxyethyl.        An “alkoxy” is intended to mean the radical —OR_(a), where R_(a)        is an alkyl group. Exemplary alkoxy groups include methoxy,        ethoxy, propoxy, and the like.

The subject invention further pertains to isolated enantiomericcompounds. The isolated enantiomeric forms of the compounds of theinvention are substantially free from one another (i.e., in enantiomericexcess). In other words, the “R” forms of the compounds aresubstantially free from the “S” forms of the compounds and are, thus, inenantiomeric excess of the “S” forms. Conversely, “S” forms of thecompounds are substantially free of “R” forms of the compounds and are,thus, in enantiomeric excess of the “R” forms. In one embodiment of theinvention, the isolated enantiomeric compounds are at least about in 80%enantiomeric excess. In a preferred embodiment, the compounds are in atleast about 90% enantiomeric excess. In a more preferred embodiment, thecompounds are in at least about 95% enantiomeric excess. In an even morepreferred embodiment, the compounds are in at least about 97.5%enantiomeric excess. In a most preferred embodiment, the compounds arein at least about 99% enantiomeric excess.

The term “subject,” as used herein, describes an organism, includingmammals such as primates, to which treatment with the compositionsaccording to the present invention can be provided. Mammalian speciesthat can benefit from the disclosed methods of treatment include, butare not limited to, apes, chimpanzees, orangutans, humans, monkeys; anddomesticated animals such as dogs, cats, horses, cattle, pigs, sheep,goats, chickens, mice, rats, guinea pigs, and hamsters.

In a specific embodiment, the subject invention pertains to a compoundreferred to herein as B22EES1-8-3 (abbreviated as B8-3), which wasidentified after 5 rounds of extraction. The structure of B8-3 is:

Advantageously, this compound inhibits TNF-α induction.

After the identification of B8-3, its biological activities werecompared to dexamethasone, the standard drug for immunosuppression.Incubation with B8-3 ameliorated the LPS-upregulated TNF-α production byover 50% (FIG. 5A), which is comparable to the effects of dexamethasone(FIG. 5B).

Dexamethasone is an effective drug used in the treatment of manyautoimmune diseases. Unfortunately, the use of dexamethasone is wellknown to have side effects to the patients. Since B8-3 is isolated fromthe herbs including Cimicifuga foetida and Cimicifuga heracleifolia, thetoxicity of the herbs in human uses has been well tested for centuries.

Furthermore, it was determined that the activation of MAP kinase andNF-κB can be abrogated by B8-3. These two mediators play a key role incytokine production and, thus, regulating multiple immune responses.B8-3 can also be used according to the subject invention to regulate thedownstream effectors of TNF-α.

B8-3 was isolated from Cimicifuga racemosa and its Chinese counterpartsusing unique isolation and bioassay-guided procedures. The effects ofB8-3 on the regulation of cytokines occur via its activity in themodulation of signaling kinase and transcription factor activities. B8-3suppresses mitogen induced inflammatory response, which makes thismolecule useful for treatment of a variety of clinical conditions. Sinceoverproduction of TNF-α is toxic and can result in severe complications,limiting the overwhelming inflammatory response can be beneficial topatients in clinical management. This is the first study to identify anactive anti-inflammatory compound in Cimicifuga racemosa and its Chinesecounterparts. The compounds of the subject invention can also be used totreat inflammation associated with infection, including, but not limitedto, infections by viruses, bacteria, fungi, yeast, and other microbes.Additionally, the compounds of the subject invention can be used totreat inflammation mediated by a variety of factors including, but notlimited to, interferons, interleukins, and environmental toxins.

The compounds and pharmaceutical compositions of the present inventioncan be used in the treatment, or amelioration, of inflammatory symptomsin any disease, condition or disorder where immune and/or inflammationsuppression is beneficial. Inflammatory diseases, conditions ordisorders in which the compounds and compositions of the presentinvention can be used to inhibit unwanted immune reactions andinflammation include, but are not limited to, arthritis, including butnot limited to rheumatoid arthritis, and other diseases, conditions ordisorders of the joints or musculoskeletal system in which immune and/orinflammation suppression is beneficial.

Moreover, the compounds and compositions are also useful to treat orameliorate inflammation associated with atherosclerosis;arteriosclerosis; atherosclerotic heart disease; reperfusion injury;cardiac arrest; myocardial infarction; vascular inflammatory disordersincluding cerebro-vascular disease (stroke); respiratory distresssyndrome and other cardiopulmonary diseases, conditions or disorderswhere immune and/or inflammation suppression would be beneficial.

In addition, the compounds and compositions are also useful to treat orameliorate inflammation associated with peptic ulcer; ulcerativecolitis, Crohn's Disease, irritable bowel syndrome, other inflammatorybowel conditions, and other diseases, conditions or disorders of thegastrointestinal tract where immune inflammation suppression would bebeneficial; hepatic fibrosis; liver cirrhosis and other hepaticdiseases, conditions or disorders where immune and/or inflammationsuppression would be beneficial; thyroiditis and other glandulardiseases, conditions or disorders where immune and/or inflammationsuppression would be beneficial; glomerulonephritis and other renal andurologic diseases, conditions or disorders where immune and/orinflammation suppression would be beneficial.

In addition, the compounds and compositions are also useful to treat orameliorate inflammation associated with post-traumatic inflammation;septic shock; infectious diseases where immune and/or inflammationsuppression would be beneficial; inflammatory complications and sideeffects of surgery where immune and/or inflammation suppression would bebeneficial; bone marrow transplantation and other transplantationcomplications and/or side effects where immune and/or inflammationsuppression would be beneficial; inflammatory and/or immunecomplications and side effects of gene therapy, e.g., due to infectionwith a viral carrier; and inflammation associated with acquired immunedeficiency syndrome (AIDS).

Further, the compounds and compositions are also useful to inhibitmacrophage or T cell associated aspects of an immune response that arenot associated with inflammation. The compounds and compositions areable to inhibit macrophage or T cell activities including, but notlimited to, macrophage antigen-presenting activity, macrophage cytokineproduction, T cell cytokine production, T cell adhesion activity, T cellproliferation, etc. Thus, the peptides, peptide derivatives andcompositions are useful to suppress or inhibit a humoral and/or cellularimmune response.

The compounds and compositions are also useful to treat or amelioratemonocyte and leukocyte proliferative diseases, e.g., leukemia, byreducing the amount of monocytes and lymphocytes.

The compounds and pharmaceutical compositions of the invention arefurther useful for the prevention and/or treatment of graft rejection incases of transplantation of natural or artificial cells, tissue andorgans, such as cornea, bone marrow, organs, lenses, pacemakers, naturaland artificial skin tissue, and the like.

The compounds and compositions are also useful to treat or ameliorateinflammation associated with hypersensitivity; allergic reactions;asthma; systemic lupus erythematosus; collagen diseases and otherautoimmune diseases, conditions or disorders in which immune and/orinflammation suppression is beneficial.

The compounds and compositions are also useful to treat or ameliorateinflammation associated with otitis and other otorhinolaryngologicaldiseases, conditions or disorders where immune and/or inflammationsuppression would be beneficial; dermatitis and other dermal diseases,conditions or disorders where immune and/or inflammation suppressionwould be beneficial; periodontal diseases and other dental diseases,conditions or disorders where immune and/or inflammation suppressionwould be beneficial.

In addition, the compounds and compositions are also useful to treat orameliorate inflammation associated with posterior uveitis; intermediateuveitis; anterior uveitis; conjunctivitis; chorioretinitis;uveoretinitis; optic neuritis; intraocular inflammation, such asretinitis and cystoid macular edema; sympathetic ophthalmia; scleritis;retinitis pigmentosa; immune and inflammatory components of degenerativefondus disease; inflammatory components of ocular trauma; ocularinflammation caused by infection; proliferative vitreoretinopathies;acute ischemic optic neuropathy; excessive scarring, for example,following glaucoma filtration operation; immune and/or inflammationreaction against ocular implants and other immune andinflammatory-related ophthalmic diseases, conditions or disorders whereimmune and/or inflammation suppression would be beneficial.

Moreover, the compounds and compositions are also useful to treat orameliorate inflammation associated with autoimmune diseases andconditions or disorders where, both in the central nervous system (CNS)and in any other organ, immune and/or inflammation suppression would bebeneficial; Parkinson's disease; complications and/or side effects fromtreatment of Parkinson's disease; AIDS-related dementia complex(HIV-related encephalopathy); Devic's disease; Sydenham chorea;Alzheimer's disease and other degenerative diseases, conditions ordisorders of the central nervous system where immune and/or inflammationsuppression would be beneficial; inflammatory components of strokes;post-polio syndrome; immune and inflammatory components of psychiatricdisorders; myelitis; encephalitis; subacute sclerosing panencephalitis;encephalomyelitis; acute neuropathy; subacute neuropathy; chronicneuropathy; Guillaim-Barre syndrome; Sydenham chorea; myasthenia gravis;pseudotumor cerebri; Down's Syndrome; Huntington's disease; amyotrophiclateral sclerosis; inflammatory components of central nervous system(CNS) compression or CNS trauma or cerebrovascular accidents (stroke) orinfections or hypoxia-ischemia of the CNS; inflammatory components ofmuscular atrophies and dystrophies; and immune and inflammatory relateddiseases, conditions or disorders of the central and peripheral nervoussystems where immune and/or inflammation suppression would bebeneficial.

In yet another embodiment, the compounds and compositions of theinvention are useful to restore immune privilege at an immune privilegedsite which has lost its immune privilege such as brain, eye and testis.

In one embodiment, the subject invention provides isolated compounds. Asused herein, “isolated” refers to compounds that have been removed fromany environment in which they may exist in nature. For example, isolatedB8-3 would not refer to the B8-3 compound as it exists in C. racemosa.In preferred embodiments, the compounds of the subject invention are atleast 75% pure, preferably at least 90% pure, more preferably are morethan 95% pure, and most preferably are more than 99% pure (substantiallypure).

The present invention also provides for therapeutic or pharmaceuticalcompositions comprising a compound of the invention in a form that canbe combined with a pharmaceutically acceptable carrier. In this context,the compound may be, for example, isolated or substantially pure. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the compound is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleumoil such as mineral oil, vegetable oil such as peanut oil, soybean oil,and sesame oil, animal oil, or oil of synthetic origin. Saline solutionsand aqueous dextrose and glycerol solutions can also be employed asliquid carriers, particularly for injectable solutions. Particularlypreferred pharmaceutical carriers for treatment of or amelioration ofinflammation in the central nervous system are carriers that canpenetrate the blood/brain barrier. As used herein carriers do notinclude the natural plant material as it exists in nature.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The therapeuticcomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsion, tablets, capsules,powders, sustained-release formulations and the like. The compositioncan be formulated with traditional binders and carriers such astriglycerides. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchcompositions contain a therapeutically effective amount of thetherapeutic composition, together with a suitable amount of carrier soas to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted for localinjection administration to human beings. Typically, compositions forlocal injection administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients may be mixed prior to administration.

The therapeutic or pharmaceutical compositions of the invention can beformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with free amino groups such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with free carboxyl groups such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The present invention also provides for the modification of the compoundsuch that it is more stable once administered to a subject, i.e., onceadministered it has a longer time period of effectiveness as compared tothe unmodified compound. Such modifications are well known to those ofskill in the art, e.g., polyethylene glycol derivatization (PEGylation),microencapsulation, etc.

The amount of the therapeutic or pharmaceutical composition of theinvention which is effective in the treatment of a particular disease,condition or disorder will depend on the nature of the disease,condition or disorder and can be determined by standard clinicaltechniques. In general, the dosage ranges from about 0.001 mg/kg toabout 2 mg/kg. In addition, in vitro assays may optionally be employedto help identify optimal dosage ranges. The precise dose to be employedin the formulation will also depend on the route of administration, andthe seriousness of the disease, condition or disorder, and should bedecided according to the judgment of the practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. For example,in order to obtain an effective mg/kg dose for humans based on datagenerated from rat studies, the effective mg/kg dosage in rats isdivided by six.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients, e.g.,compound, carrier, of the pharmaceutical compositions of the invention.

The compounds of the subject invention can also be formulated consistentwith traditional Chinese medicine practices. The composition and dosageof the formulation that are effective in the treatment of a particulardisease, condition or disorder will depend on the nature of the disease,condition or disorder by standard clinical techniques.

The traditional Chinese medicine in prescription amounts can be readilymade into any form of drug, suitable for administering to humans oranimals. Suitable forms include, for example, tinctures, decoctions, anddry extracts. These can be taken orally, applied through venousinjection or mucous membranes. The active ingredient can also beformulated into capsules, powder, pallets, pastille, suppositories, oralsolutions, pasteurized gastroenteric suspension injections, small orlarge amounts of injection, frozen power injections, pasteurized powderinjections and the like. All of the above-mentioned methods are known topeople skilled in the art, described in books and commonly used bypractitioners of herbal medicine.

A tincture is prepared by suspending herbs in a solution of alcohol,such as, for example, wine or liquor. After a period of suspension, theliquid (the alcohol solution) may been administered for example, two orthree times a day, one teaspoon each time.

A decoction is a common form of herbal preparation. It is traditionallyprepared in a clay pot, but can also be prepared in glass, enamel orstainless steel containers. The formulation can be soaked for a periodof time in water and then brought to a boil and simmered until theamount of water is reduced by, for example, half.

An extract is a concentrated preparation of the essential constituentsof a medicinal herb. Typically, the essential constituents are extractedfrom the herbs by suspending the herbs in an appropriate choice ofsolvent, typically, water, ethanol/water mixture, methanol, butanol,iso-butanol, acetone, hexane, petroleum ether or other organic solvents.The extracting process may be further facilitated by means ofmaceration, percolation, repercolation, counter-current extraction,turbo-extraction, or by carbon-dioxide hypercritical(temperature/pressure) extraction. After filtration to rid of herbdebris, the extracting solution may be further evaporated and thusconcentrated to yield a soft extract (extractum spissum) and/oreventually a dried extract, extracum siccum, by means of spray drying,vacuum oven drying, fluid-bed drying or freeze-drying. The soft extractor dried extract may be further dissolved in a suitable liquid to adesired concentration for administering or processed into a form such aspills, capsules, injections, etc.

Materials and Methods

Plant Material

Cimicifuga racemosa was purchased from the Glenbrook Farms Herbs andSuch, Campbellsville, Ky. Cimicifuga heracleifolia, Cimicifuga foetidaand Cimicifuga dahurica were purchased in herbal markets andsubsequently authenticated by Purapharm with respect to theiridentification.

Extraction and Isolation of the Bioactive Molecules

The procedures for plant extraction are shown in FIG. 1. Briefly,Cimicifuga racemosa (1.8 kg) was milled, homogenized and then suspendedin (1:5) milli-Q water for 1 hr with continuous sonication. Thesupernatant was filtered through an analytical filter paper and thenpartitioned three times with ethyl acetate (EtOAc) (1:1). The resultingEtOAc extract was concentrated to dryness in vacuo (35° C.) to yield14.97 g of a dark brown residue. The residue was reconstituted inmethanol (MeOH) and then fractionated by partitioning with hexane(n-C₆H₁₄). The MeOH fraction was concentrated and reconstituted in H₂Oand then partitioned sequentially with EtOAc and butanol (n-BuOH). Fourfractions, namely n-C₆H₁₄, EtOAc, n-BuOH, and H₂O were obtained.

The fraction that showed inhibitory effects on LPS-induced TNF-αproduction was subjected to additional silica gel 60A (35-75 μm)chromatography using n-C₆H₁₄, EtOAc, and MeOH to yield six fractions.The active fractions were further purified by reversed-phasehigh-performance liquid chromatography (HPLC) (Lichrospher 100 RP C18 EC5μ, 250×4.6 mm ID) using a gradient elution from 25% acetonitrile(CH₃CN) to 90% CH₃CN at a flow rate of 1 mL min⁻¹.

Peak detection was achieved using an Agilent 1200 series of fastscanning Photo-diode Array detector set at 254, 210 and 280 nm. Elutingpeaks were scanned between 200 nm and 300 nm with 1 nm intervals todetermine absorbance maxima and minima.

By repeating the purification process using HPLC, a single compound waseluted at approximately 9.4 minutes with UV absorbance maximized at 290and 325 nm, which revealed that it has a conjugated aromatic system.This compound (B22EES1-8-3) showed anti-inflammatory activities.

Elucidation of the Molecular Structure

The structure of the resulting pure compound (B22EES1-8-3) waselucidated by using a Bruker 500 MHz DRX NMR spectrometer, operating at500 MHz for ¹H and at 125.765 MHz for ¹³C NMR, using methanol-d as thesolvent. Distortionless enhancement by polarization transfer (DEPT)experiments were performed using a transfer pulse of 135° to obtainpositive signals for CH and CH₃, and negative signals for CH₂. HR-ESI-MSwas performed on a micrOTOF II 411 ESI-TOF mass spectrometer (BrukerDaltonics). Data sets were acquired in negative electrospray (ESI) modein a scan ranging from 100 to 1600 m/z at a sampling rate of 2 Hz. ESIparameters were as follows: capillary, 3.2 kV; nebulizer pressure, 4bar; dry 415 gas flow, 8 L/min; and dry gas temperature, 200° C.

The 13C NMR spectra of the compound showed signals at δ 68.6 (t, C-1),204.6 (s, C-2), 46.4 (t, C-3), 126.1 (s, C-4), 117.7 (d, C-5), 146.7 (s,C-6), 145.8 (s, C-7), 116.7 (d, C-8), 122.1 (d, C-9), 168.3 (s, C-1′),115.3 (d, C-2′), 147.6 (d, C-3′), 128.9 (s, C-4′), 114.9 (d, C-5′),148.2 (s, C-6′), 151.8 (s, C-7′), 112.6 (d, C-8′), 123.1 (d, C-9′), and56.5 (q, MeO-7′). In addition, the compound showed a [M]-ion peak at m/z357.0952 in its HR-ESI-MS, consistent with the molecular formulaC₁₉H₁₇O₇ (calc. 357.0974).

Determination of the Presence of B22EES1-8-3 in C. foetida and C.heracleifolia Using HPLC-UV and HPLC-TOF-MS

Herbs C. foetida and C. heracleifolia were extracted following theextraction procedure of C. racemosa as described above. The extracts ofHerbs C. foetida and C. heracleifolia (CF22EES1-8 and CH22EES1-8) wereinjected into the HPLC equipped with a PDA detector following thechromatographic conditions that were used to isolate B22EES1-8-3. Thechromatogram of individual sample was recorded. CF22EES1-8 andCH22EES1-8 were also injected separately into an Acquity UPLC system(Waters, USA) equipped with an Xterra MSC18 column (150*2.1 mmID, 3.5522μm). Chromatographic separations were performed using a gradient elutionfrom 25% acetonitrile (CH₃CN) to 90% CH₃CN at a flow rate of 200 μL/min.Eluted compounds were detected using a micrOTOF II ESI-TOF massspectrometer (Bruker Daltonics). Data sets were acquired in negativeelectro spray (ESI) mode in a scan ranging from 100 to 1600 m/z at asampling rate of 2 Hz. ESI parameters were as follows: capillary, 3.2kV; nebulizer pressure, 4 bar; dry gas flow, 8 L/min; and dry gastemperature, 200° C.

By comparing their peaks with the standard of B22EES1-8-3, the presenceof B22EES1-8-3 in the extract of C. foetida and C. heracleifolia weredetermined.

Chemicals

Endotoxin (lipopolysacharride, LPS) from E. coli was purchased fromSigma and used as an inducer of TNF-α expression. Dexamethasone (Sigma)was used as a control drug to inhibit the LPS induction of TNF-α.

Cell Culture and Primary Blood Macrophage Isolation

Human peripheral blood monocytic cells (PBMC) were isolated from thebuffy coat of healthy donor blood supplied by Hong Kong Red Cross byFicoll-Paque (Amersham Pharmacia Biotech, Piscataway, N.J.) densitygradient centrifugation as described in our previous reports^(14,15,34).In brief, the buffy coat was spun at 3000 rotations per min (rpm) for 15min to separate the blood cells and the plasma. The heat inactivatedserum was filtered for future use.

The cell layer was diluted with phosphate buffered saline (PBS) in aratio of 1:1. The diluted cells were overlaid on Ficoll-Paque slowly andcentrifuged at 2300 rpm for 20 min for separation of mononuclear cellsfrom erythrocytes. The mononuclear cell layer was removed and washedwith RPMI 1640 medium until the supernatant was clear.

The cells were finally resuspended in RPMI 1640 medium supplemented with5% autologous serum and cultured for 1 hr. The non-adherent cells wereremoved afterwards and the remaining adherent cells were furtherincubated for another 24 hr at 37° C. in 5% carbon dioxide (CO₂).

The adherent monocytic cells were detached and seeded onto tissueculture plates and incubated for another 7-14 days in order todifferentiate the primary blood monocytic cells to primary bloodmacrophages (PBMac).

Isolation of RNA and Reverse Transcription

Total RNA from primary blood macrophages with or without treatment ofCimicifuga racemosa fractions was extracted by TRIzol (Invitrogen).Reverse transcription (RT) of messenger RNA (mRNA) to complementary DNA(cDNA) was done by using the SuperScript II system (Invitrogen) as perthe manufacturer's instruction.

Polymerase Chain Reaction (PCR) and Real-Time RT-PCR

Semi-quantitative PCR assays of targeted genes were performed in a 25 μlreaction mixture containing 1.5 mM MgCl₂, 0.2 mM of each deoxynucleosidetriphosphate, 0.25 μM of each primer, 2 units of Taq polymerase(Amersham Pharmacia Biotech, Piscataway, N.J.), and 1 μl of cDNA. PCRprimer sets for TNF-α and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were as follows. TNF-α (upstream: 5′-GGCTCCAGGCGGTGCT TGTCC-3′(SEQ ID NO:1); downstream: 5′-AGACGGCGATGCGGCTGATG-3′ (SEQ ID NO:2)),and GAPDH (upstream: 5′-ACCACAGTCCATGCCATCAC-3′ (SEQ ID NO:3);downstream: 5′-TCCACCACCCTGTTGCTGTA-3′ (SEQ ID NO:4). The thermalcycling condition for PCR was 94° C. for 30 s, 60° C. for 30 s, and 72°C. for 1 min. The cycling reactions were repeated for 24 more cycles.

Quantitative RT-PCR was performed according to the manufacturer'sinstructions by using Applied Biosystems TaqMan® Universal Master Mix.The TNF-α TaqMan probes was purchased from the Applied Biosystems, and18s RNA was used as an internal control. Samples were allowed to run intriplicates in each Quantitative RT-PCR assay.

Enzyme-Linked ImmunoSorbent Assay (ELISA)

Culture supernatants of the LPS-treated PBMac, with or withoutB22EES1-8-3 pretreatment, were collected at different time intervals andstored at −70° C. The levels of the secreted TNF-α were measured byELISA kits specific for the cytokine (R&D system, Minneapolis, Minn.).

Preparation of Cellular Extracts

For the collection of whole cell lysate, PBMac were washed with cold PBSand incubated in cold lysis buffer (50 mMtris(hydroxymethyl)aminomethane-chloride (Tris-Cl) [pH7.4]; 150 mMsodium chloride (NaCl); 50 mM sodium fluoride (NaF); 10 mMβ-glycerophosphate; 0.1 mM ethylenediaminetetraacetic acid (EDTA); 10%glycerol; 1% Triton X-100; 1 mM phenylmethanesulphonylfluoride (PMSF); 1mM sodium orthovanadate; 2 μg/mL pepstatin A; 2 μg/mL aprotinin and 2μg/mL leupeptin) for 20 min. The lysate was then centrifuged at 4° C.for 20 min. The supernatant was collected and stored at −70° C. untiluse.

To collect nuclear protein extracts, the treated cells were washed withPBS and resuspended in buffer A (10 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) [pH7.9], 10mM potassium chloride (KCl), 0.1 mM EDTA, 0.1 mM ethylene glycoltetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 0.5 mMphenylmethanesulphonylfluoride or phenylmethylsulphonyl fluoride (PMSF),2 μg aprotinin, 1 mM sodium orthovanadate, 2 μg/mL pepstatin A, 2 μg/mLleupeptin and 50 mM NaF) for 15 min. After that, NP-40 at a finalconcentration of 0.625% was added and mixed vigorously for cell lysis.

The cell lysate was centrifuged and the supernatant containingcytoplasmic proteins was collected for storage at −70° C. The nuclearpellet was resuspended in buffer C (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF) for 15 min on ice tocomplete lysis of the nuclear membrane. The nuclear lysate was thencentrifuged, and the supernatant containing the nuclear protein wascollected and stored at −70° C.^(34,35).

Western Blot Analysis

Whole cell lysate (20 μg) or nuclear protein (2 μg) were separated bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andtransferred to nitrocellulose membranes for probing overnight with therespective antibodies specific for the phosphorylated or total form ofERK1/2 and p38 MAPK (Cell Signaling Technology, Beverly, Mass.), NF-κBp65 protein and lamin B (Santa Cruz Biotechnology, Santa Cruz, Calif.).The membranes were incubated with the corresponding secondary antibodiesconjugated with horseradish peroxidase (BD Transduction Lab, San Diego,Calif.). The signal was visualized by using enhanced chemiluminescencekit (Amersham Pharmacia Biotech). In order to quantify the results fromthe Western blots, the gels were scanned and the intensity of the bandswas analyzed by a computer program Quantity One from BioRad.

The scope of the invention is not limited by the specific examples andsuggested procedures and uses related herein since modifications can bemade within such scope from the information provided by thisspecification to those skilled in the art.

A more complete understanding of the invention can be obtained byreference to the following specific examples of compounds, compositions,and methods of the invention. The following examples illustrateprocedures for practicing the invention. These examples should not beconstrued as limiting. It will be apparent to those skilled in the artthat the examples involve use of materials and reagents that arecommercially available from known sources, e.g., chemical supply houses,so no details are given respecting them.

EXAMPLE 1 Extraction and Identification of B22EES1-8-3

A light brown powder was obtained by repeated partitioning of the EtOAcfraction prepared from the rhizomes of Cimicifuga racemosa andsequential chromatography on silica gel and reversed-phase HPLC. Thedetailed procedures are summarized in FIG. 1.

Using HPLC, the compound was eluted at approximate 9.4 min as a singlecompound with UV absorbance at wavelength 254, 210 and 280 nm (FIG. 2A).In FIG. 2B, the UV absorbance of the compound maximized at 290 and 325nm, which revealed that it has a conjugated aromatic system. Thecompound showed a [M]⁻ ion peak at m/z 357.0952 in its HR-ESI-MS.Together with the ¹H and ¹³C spectra data (FIG. 3), it was elucidated asB22EES1-8-3.

EXAMPLE 2 Bio-Assays

The chemical compound in Cimicifuga racemosa responsible for theinhibition of LPS-induced expression of TNF-α was identified. LPS iswell known to be a potent inducer of TNF-α and its effects cannot beeasily suppressed without the use of cytotoxic agents.

Bacterial endotoxin (lipopolysaccharide, LPS) stimulation of TNF-αinduction in primary macrophages was used as a model of inflammatorydiseases, since the production of TNF-α is an indicator of a key immuneresponse.

Individual extracts isolated from Cimicifuga racemosa were incubatedwith PBMac for 24 hr prior to the addition of LPS for another 3 hr.Total RNA of the treated samples was isolated and subjected to RT-PCRassays using specific human TNF-α primers. The results showed that thefraction B22EES1 inhibits LPS-induced TNF-α mRNA expression (FIG. 4A,lanes 2 and 4). Among the sub-fractions of B22EES1, only B22EES1-4 andB22EES1-8 retained the suppressive activities for TNF-α induction (FIG.4A, lanes 12 and 20).

EXAMPLE 3 Effects of B22EES1-8-3 on LPS-Induced cytokine Production

After the identification of B22EES1-8 as being responsible for theinhibitory effects on TNF-α, the activities of B22EES1-8 sub-fractionsas described above were separated and analyzed. A single molecule,namely B22EES1-8-3 (abbreviated as B8-3), was found to be the activecompound in the herbal extract responsible for the anti-inflammatoryeffects.

To confirm the activities of B8-3 in suppressing TNF-α production, B8-3was incubated with PBMac for 24 hr prior the addition of LPS atconcentrations of 1 ng/mL and 10 ng/mL for 24 hr. The culturesupernatants were collected and measured by ELISA for the level ofsecreted TNF-α.

B8-3 inhibited the LPS-induced TNF-α protein production by 47±19% and58±30% at LPS concentrations of 1 ng/mL and 10 ng/mL, respectively (FIG.5A, lanes 4 vs 5 and lanes 6 vs 7).

To further compare the efficiency of B8-3 with existing drugs,dexamethasone, a potent immunosuppressive corticosteroid, was used as aprototype. PBMac were treated with dexamethasone for 24 hr prior to theaddition of LPS at concentrations of 1 ng/mL and 10 ng/mL for 24 hr.

The results demonstrate that dexamethasone causes a significantinhibition of LPS-induced TNF-α production by 32±7.5% and 25±6.3% atconcentrations of 1.3 and 5.1 μM, respectively (FIG. 5B).

EXAMPLE 4 Molecular Mechanisms of Cytokine Downregulation by B8-3

The molecular pathways involved in B8-3 inhibition of LPS-induced TNF-αproduction were elucidated. It is well documented that the activation ofcytokine production in LPS-treated cells is initiated by the binding ofLPS to its receptor³⁸. After binding to the receptor, a cascade ofsignaling kinases is activated. Among the activated kinases, MAP kinasesplay a crucial role in LPS-induced cytokine production. Previous studiesillustrated that the induction of TNF-α by LPS and other pathogensrequires the phosphorylation and activation of ERK1/2 and p38MAPK^(13,14,39).

In order to study the role of MAP kinases in B8-3 inhibition of TNF-αproduction, PBMac were treated with B8-3 for 24 hr and followed by theaddition of LPS for 15 min. Protein samples were collected afterward andWestern blots were performed.

The results showed that LPS treatment results in phosphorylation of twodifferent MAP kinases, namely ERK1/2 and p38 MAPK (FIG. 6. lane 2). WithB8-3 pretreatment, the phosphorylation of ERK1/2 (FIG. 6A, lanes 2 vs 4)but not p38 MAPK induced by LPS was suppressed (FIG. 6B, lanes 2 vs 4).

These results demonstrated that the anti-inflammatory activity of B8-3may be in part due to its inhibition of ERK1/2 phosphorylation.

Along the signaling pathways regulated by MAP kinases in response to LPStreatment, activation of the transcription factor NF-κB plays a criticalrole in the induction of proinflammatory cytokines including TNF-α⁴⁰.The activation of NF-κB involves degradation of its specific inhibitorIκB and translocation of NF-κB sub-units from the cytoplasm to thenucleus. In accordance with the subject invention, the addition of B8-3for 24 hr prior the addition of LPS reduced the translocation of NF-κBp65 subunit into the nucleus.

The results showed that the addition of B8-3 to PBMac for 24 hr prior tothe addition of LPS reduced the amount of p65NF-kB in the nuclearfraction (FIG. 6C, lanes 2 vs 4), indicating that the translocation ofthe p65NF-kB to the nucleus was inhibited by B8-3. In general, B8-3 caninhibit LPS-induced kinase activities and their consequent activation ofthe nuclear transcription factor for TNF-α transcription. Thus, thecompounds of the subject invention can be used to regulate intracellularand/or extracellular activities that are downstream from NF-kB and/orERK1/2 in the cascade of cellular events associated with inflammatoryconditions.

EXAMPLE 5 Determination the Presence of B22EES1-8-3 in Cimicifugafoetida and Cimicifuga heracleifolia Using HPLC-UV

Under the same HPLC conditions, the retention time and the UV absorbanceof B8-3 were compared with the characteristic peak in the chromatogramsof CF22EES1 and CH22EES1-8. In FIGS. 7A and B, both samples had a peakwith retention time at approximate 9.4 min and their respective UVabsorbance was same as that of B8-3 (FIGS. 2A & B). The results revealedthat herbs including C. foetida and C. heracleifolia contained B8-3.

EXAMPLE 6 Determination the Presence of B22EES1-8-3 in Cimicifugafoetida and Cimicifuga heracleifolia Using HPLC-TOF-MS

Under the same HPLC and ESI-MS conditions, the retention time and themass-to-charge ratio of B8-3 were compared to the characteristic peak inthe chromatograms and spectra of CF22EES1-8 and CH22EES1-8. In FIGS. 8Band C, both samples had a peak with retention time at approximate 6 minwith an ion peak at m/z 357 that was the same as that of compound 1(FIG. 8A). The results revealed that herbs including C. foetida and C.heracleifolia contained B8-3.

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1. A method for treating arthritis, wherein said method comprisesadministering, to a subject in need of such treatment, an effectiveamount of an isolated compound or a salt thereof, wherein said compoundhas the following formula:

wherein R₁ is alkyl; R₂ is H or alkyl; R₃, R₄, and R₅ are independently—H, acyl, halo, haloalkyl, amino, alkylamino, hydroxyl, alkyl,hydroxylalkyl, or —COOH; R₆ is —O or —NH; R₇ is —H, alkyl, alkoxy,hydroxylalkyl, hydroxyl, or halo; R₈, R₉, and R₁₂ are independently —H,acyl, halo, amino, alkylamino, hydroxyl, alkyl, hydroxylalkyl, or —COOH;R₁₀ is H or alkyl; and R₁₁ is H or alkyl.
 2. The method, according toclaim 1, wherein the subject is a human.
 3. The method, according toclaim 1, wherein the R₂ is H, R₃ is H, and R₄ is H.
 4. The method,according to claim 3, wherein R₁ is a methyl group.
 5. The method,according to claim 1, wherein TNF-α activity is inhibited.
 6. Themethod, according to claim 1, used to rheumatoid arthritis.
 7. Themethod, according to claim 1, wherein the subject is a mammal.
 8. Themethod, according to claim 7, wherein the mammal is cattle.
 9. Themethod, according to claim 1, wherein ERK1/2 phosphorylation isinhibited.
 10. The method, according to claim 1, wherein said compoundis:


11. The method, according to claim 1, wherein NF-kB activation issuppressed.